PROJECT SUMMARY After several years of research on biological therapies and small molecules to target inflammation, we need a different strategy to further get more insights into mechanisms underlying RA pathogenesis and identify potential new treatments, as a significant proportion of patients are partial responders. In other fields such as oncology the concept of metabolic reprograming to improve immunotherapy are concepts we truly believe should be translated into autoimmune diseases to complement current therapies. However, there are little data about targeting metabolic changes in RA. We seek with this grant a better understanding of the biology of these metabolic pathways in RA to better characterize a new approach in its therapeutic armamentarium. Our finding that hexokinase 2 (HK2) activity is enhanced only in RA synovium and in different RA synovial cells, suggest a cooperative metabolic reprograming in the joint that contributes to RA development and progression. Our preliminary data demonstrate that while HK1 expression is expressed in both OA and RA synovium, HK2 expression co-localizes with MO and FLS markers, and is only observed in RA and not in OA synovial samples. We also show that HK2 regulates key FLS function as HK2 knockdown impaired FLS invasion. Conversely, HK2 overexpression increases FLS invasion and migration rate. Of note, lactate and PLOD2, which are involved in cell migration and invasion, are upregulated after HK2 expression. Up-regulation of extracellular lactate also suggests a metabolic shift towards accelerated glycolytic metabolism. An HK2 mutant lacking its mitochondrial- binding motif (HK2?N) reversed the invasive phenotype. In MO, a peptide that dissociates HK2 from mitochondria, impaired IL-6 secretion. Importantly, adenovirus-mediated expression of HK2 in the knee by intra- articular injection induced synovial thickness, which was much less evident when HK2?N was intra-articular injected. Finally, HK2F/F-Col1a1 mice, which deletes HK2 in FLS among other non-hematopoietic cells, and treatment with clotrimazole, which dissociated HK2 from mitochondria, significantly decreased arthritis severity. Thus, we will test the hypothesis that mitochondrial HK2 is key regulator of FLS phenotype and MO activation, which contributes to joint destruction in RA. The identification of HK2, an isoform-specific contributor to elevated cell glucose metabolism in RA synovial tissue offers a safer approach than global glycolysis inhibition. HK2 could be selectively targeted without compromising systemic homeostasis or corresponding metabolic function in normal cells as a novel additional approach for combination therapy in RA joint disease independent of systemic immunosuppression.